Caso 1: Pol

In spite of the plethora of plant metabolites with tumor cytotoxic or immuno-stimulating properties, plants may not be an ideal source of natural anticancer drugs. There are a number of reasons that make the recovery of plant-derived products less attractive than the alternative methods of biotechnology and chemical synthesis.

First of all, there is the supply issue. In several cases, compounds of interest come from slow growing plant species (e.g. woody species), or species that are endangered. In addition, in vivo productivity can be considerably low, thus necessitating the use of an overwhelming amount of plant biomass in order to obtain a satisfactory portion of the natural product (especially in the case of secondary metabolites). For example, taxol concentration in needles and dried bark of Taxus brevifolia is approx. 0.01–0.1%. Supply can also be hindered due to inadequate plant pro-duction, which, in turn, may be caused by a number of problems related to disease, drought or socioeconomic factors. As demonstrated previously for taxol the issue of supply can be partly resolved by breeding for high-yielding varieties, introduction of wild species or derivation (in abundant amounts) of precursors for the hemisynthesis of the desired product (Cragg et al., 1993; Gragg, 1998).

Second, plant-derived pharmaceutical extracts frequently lack the necessary standardization that could render them reliable for large-scale, clinical use. This problem is related to a number of factors, such as the dependence of the production on environmental factors and the plant developmental stage (e.g. flowering), as well as the heterogeneity of the extracts, which makes further isolation and purification of the product an indispensable, though costly step.

A representative example is mistletoe lectin extract, which presents a remarkable seasonal variation in the levels of ML isolectins (ML I, ML II and ML III). Furthermore, the specific bioactivity of the extract fluctuates over a prolonged (e.g. two-year) storage time (Lorch and Troger, 2000).

Biotechnology could offer an alternative method for the production of considerable plant biomass or natural products in a relatively short time (Payne et al., 1991; Kintzios and Barberaki, 2000). In vitro techniques are a major component of plant biotechnology, since they permit artificial control of several of the parameters affecting the growth and metabolism of cul-tured tissues. Plainly put, plant tissue culture works on the principle of inoculating an explant (that is a piece of plant tissue, such as a leaf or stem segment) from a donor plant on a medium containing nutrients and growth regulators and causing thereof the formation of a more or less dedifferentiated, rapidly growing callus tissue. Production of plant-derived anticancer agents could be advantageous over derivation from plants in vivo since:

1 By altering the culture parameters, it might be possible to control the quantity, composi-tion and timing of produccomposi-tion of mistletoe extracts. In this way, problems associated with the standardization of plant extracts could be overcome.

2 By feeding cultures with precursor substances for the biosynthesis of certain metabolites, a higher productivity can be achieved from cultured cells (in vitro) than from whole plants.

3 Potentially, entirely novel substances can be synthesized through biotransformation or by taking advantage of somaclonal variation, that is, a transient or heritable variability of metabolic procedures induced by the procedure of in vitro culture.

4 The establishment of a callus culture is the first step required in order to obtain genetically modified cells or plants, for example, crop plants able to specifically produce a desired product in excessive amount.

5 Protoplasts are plant cells having their cell wall artificially removed. In this way, they can be used in gene transfer experiments and for the creation of hybrid cells that result from the direct fusion of two protoplast cells that might have been derived from entirely different species.

6 Plant species that are difficult to propagate (such as mistletoe, which is exclusively accom-plished with the aid of birds, carrying distantly mistletoe seeds) could be clonally micro-propagated, thus obtaining thousands of seedlings from a very limited mass of donor tissue (essentially from one donor plant only). This can be achieved by plant regeneration via organogenesis (induction of shoots and roots from callus cultures) or somatic embryogenesis (the process of embryo formation from somatic (sporophytic) tissues without fertilization).

Promising as the perspectives of plant cell culture may be, established plant-derived commercial anticancer drugs (such as vinblastine and vincristine from Catharanthus roseus) are still produced by isolation from growing plants; eventually drugs are semisynthetically produced from natural precursors also isolated from plant sources in vivo. Currently, there are only a few plant-derived natural compounds with antineoplastic properties that are being produced biotechnologically, mostly on the laboratory level:

Periwinkle (Vinca rosea or Catharanthus roseus): Numerous studies have been conducted on the scale-up indole alkaloid production from cell suspension cultures of C. roseus. Several factors affecting production have been evaluated, including medium nutrient and growth regulator composition, elicitors, osmotic stress and precursor (tryptophan) feeding. Vinblatine, an antileukemic dimeric indole alkaloid dimmer cannot be directly produced from C. roseus in vitro, due to under-expression of the enzyme acetyl CoA:deacetylvindoline O-acetyl transferase, which catalyzes the formation of vindoline, one of the substrates leading to anhydrovinblastine. Yield values of catharanthine (the second substrate for vinblastine synthesis) up to 17gl^1 after fungal induction have been reported (Bhadra et al., 1993).

Pacific Yew (Taxus brevifolia): In 1977, NCI awarded contracts for the investigation of plant tissue culture as a source of anticancer drugs, and two of these studies related to taxol produc-tion. Unfortunately, these contracts were terminated in 1980 before any positive results had been obtained. Considerable research effort has once more been focused on the application of this technology to taxol production. Ketchum et al. (1999) reported the production of up to 1.17%

of paclitaxel within five days of elicitation with methyl jasmonate, along with other taxoids, such as 13-acetyl-9-dihydrobaccatin, 9-dihydrobaccatin III and baccatin VI. Two companies (ESCAgenetics Corporation and Phyton Catalytic) reported on their plans for a scale-up production of taxol in the near future.

American mandrake (Podophyllum hexandrum): Cell suspensions of P. hexandrum have been established which accumulate up to 0.1% podophyllotoxin, a cytotoxic lignan used for the hemisynthesis of etoposide and teniposide. Accumulation of podophyllotoxin has been increased twelve-fold after precursor feeding with coniferin, a glucosylated intermediate of the phenyl-propanoid pathway (Smollny et al., 1998).

Mistletoe (Viscum album L.): Becker and Schwarz (1971) were the first to mention the possi-ble use of mistletoe callus cultures as a source of bioactive products. In 1990, Fukui et al.

reported on the induction of callus from leaves of V. album var. lutescens: they were able to iden-tify in the callus two galactose-binding lectins which were originally observed in mistletoe leaves.

Kintzios and Barberaki (2000) succeeded in inducing callus and protoplast cultures from mistle-toe leaves and stems in a large number of different growth regulator and media treatments.

They have also studied the effects of different plant parts (stems and leaves), harvest time (winter or summer), explant disinfection methods, growth regulators, culture medium composi-tion and cell wall digescomposi-tion treatments. Finally, they observed a relatively low (8%) somaclonal variation, in the aspect of both the quantitative and the qualitative mistletoe protein production in vitro (Kintzios and Barberaki, 2000; Kintzios et al., 2002). Langer et al. (1997) cloned different fragments of the ML gene from mistletoe genomic DNA, constructed expression vectors (A- and B-chain coding region) and the single chains were expressed in E. coli separately. Experimental investigations on the activity of recombinant mistletoe lectin (rML) were promising.